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SRP056004
Mus musculus
5 Downloadable Samples
Illumina HiSeq 2000
Description
A comprehensive landscape of epigenomic events regulated by the Reelin signaling through activation of specific cohort of cis-regulatory enhancer elements (LRN-enhancers), which involves the proteolytical processing of the LRP8 receptor by the gamma-secretase activity and is required for learning and memory behavior Overall design: All RNA-Seq experiments were designed to evaluate the transcriptional program regulated by the Reelin-LRP8 signaling pathway in neuronal cells
Publication Title
LRP8-Reelin-Regulated Neuronal Enhancer Signature Underlying Learning and Memory Formation.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 2 Downloadable Samples
- Illumina HiSeq 2000
Description
Although liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program. Overall design: RNA-sequencing of polyA selected RNA molecules in NTera2/D1 cells and Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq).
Publication Title
DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Homo sapiens
- 4 Downloadable Samples
Description
The association between hyper-inflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, non-transcriptional role of GPS2 as a guardian against hyperstimulation of TNFA-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNFA target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the non-transcriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis. Overall design: RNA-sequencing of polyA selected RNA molecules in 293T cells and ChIP-seq of GPS2, TBL1, and NCOR.
Publication Title
A protective strategy against hyperinflammatory responses requiring the nontranscriptional actions of GPS2.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
Illumina human-6 v2.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 12 Downloadable Samples
- Illumina human-6 v2.0 expression beadchip
Description
Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell type-specific gene expression programs; however, the potential that there are pre-established enhancers in different functional classes that permit alternative signal-dependent transcriptional responses has remained unexplored. Here we present evidence that cell lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), acting at structurally- and functionally-distinct classes of pre-established enhancers, thus licensing specific signal-activated responses while restricting others. Consequently, FoxA1 down-regulation, an unfavorable prognostic sign in advanced prostate tumors, causes a massive switch in AR binding from one functional class of enhancers to another, with a parallel switch in levels of enhancer-templated non-coding RNAs (eRNAs) revealed by the global run-on assay (GRO-seq), which documents the dramatic reprogramming of the hormonal response. The molecular basis for this switch lies in the release of FoxA1-mediated restriction of AR binding to the new enhancer class with no apparent nucleosome remodeling, which is required for stimulating their eRNA transcription and/or enhancing enhancer:promoter looping and gene activation. Together, these findings reveal a large repository of pre-determined enhancers in the human genome that can be dynamically tuned to induce their transcription and activation of alternative gene expression programs, which may underlie many sequential gene expression events in development or during disease progression.
Publication Title
Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 8 Downloadable Samples
- IlluminaHiSeq2000
Description
An emerging theme of gene regulation is the involvement of architectural chromosomal molecules in transcription control. Condensins are critical regulators of mitotic chromosomes, but their interphase chromatin localization and functions remain poorly understood. Here we report that both the condensin I and condensin II complexes exhibit an unexpected, dramatic 17-?-estradiol-induced preferential recruitment to oestrogen receptor ? (ER-?)-bound active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-? distinct from classic cofactors. Condensins prove to positively regulate ligand-dependent gene and eRNA transcription by modulating a binding equilibrium of enhancer-associated coactivators/corepressors, including p300 and RIP140. This activity was achieved by the condensin-dependent recruitment of an E3 ubiquitin ligase, HECTD1, to active enhancers, where it polyubiquitinates and dismisses corepressor RIP140 to stimulate eRNA transcription. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating enhancer activation, providing new insights into enhancer function in the regulated transcriptional programs Overall design: The GRO-seq measures the trancription of nascent RNAs in the genome. From MCF7 cells treated with veichle or estrodial, we could identify estrogen-regulated eRNAs and subsequently could study their functions.
Publication Title
Condensin I and II Complexes License Full Estrogen Receptor α-Dependent Enhancer Activation.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
While thousands of long non-coding RNAs (lncRNAs) are expressed in higher eukaryotes, the potential regulatory roles of lncRNAs in regulated gene transcription programs remain rather poorly understood. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the androgen receptor (AR) and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM, to the N-terminally methylated AR. Unexpectedly, recognition of the H3K4me3 promoter mark by the PHD finger-domain of Pygopus2, recruited by PCGEM1, proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells, revealing a novel aspect of ligand-induced enhancer-promoter interactions. In “resistant” prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for the robust activation of both truncated and full length AR, causing DHT-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA)-mediated targeting of these lncRNAs in these resistant cancer cell lines strongly suppressed xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors. Overall design: Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq); after knocking-down lincRNAs PCGEM1 and PRNCR1. LNCaP cells were grown to 30-50% confluence and siRNA/ASO transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Control samples were transfected with scramble ASO and control siRNA, respectively. On the following day of transfection, the cells were cultured in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM for 1 hour Scramble ASO, -DHT Scramble ASO, +DHT PRNCR1 ASO, -DHT PRNCR1 ASO, +DHT PCGEM1 ASO, -DHT PCGEM1 ASO, +DHT
Publication Title
lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 4 Downloadable Samples
- Illumina HiSeq 2000
Description
While thousands of long non-coding RNAs (lncRNAs) are expressed in higher eukaryotes, the potential regulatory roles of lncRNAs in regulated gene transcription programs remain rather poorly understood. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the androgen receptor (AR) and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM, to the N-terminally methylated AR. Unexpectedly, recognition of the H3K4me3 promoter mark by the PHD finger-domain of Pygopus2, recruited by PCGEM1, proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells, revealing a novel aspect of ligand-induced enhancer-promoter interactions. In “resistant” prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for the robust activation of both truncated and full length AR, causing DHT-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA)-mediated targeting of these lncRNAs in these resistant cancer cell lines strongly suppressed xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors. Overall design: Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq); after knocking down PYGO2 LNCaP cells were grown to 30-50% confluence and siRNA/ASO transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Control samples were transfected with scramble ASO and control siRNA, respectively. On the following day of transfection, the cells were cultured in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM for 1 hour Control siRNA, -DHT Control siRNA, +DHT Pygo2 siRNA, -DHT Pygo2 siRNA, +DHT
Publication Title
lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 4 Downloadable Samples
- Illumina HiSeq 2000
Description
Substantial evidence supports the hypothesis that enhancers are critical regulators of cell type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of the LIM domain binding protein, LDB1/CLIM2/NLI, interacting with the enhancer binding protein, ASCL1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target target gene promoter genes. While LDB1-dependend activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of MTA2, a component of the NuRD complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type. Overall design: Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq)
Publication Title
Enhancer-bound LDB1 regulates a corticotrope promoter-pausing repression program.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 4 Downloadable Samples
- IlluminaHiSeq2000
Description
Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as SD70, initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-seq-like approach, referred to as Drug-seq, we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely co-localized with androgen receptor (AR) on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the jumonji (JMJ) domain-containing demethylase, KDM4C. Drug-seq represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies. Overall design: Global Run-On (GRO) assay followed by high-throughput sequencing (GRO-seq).
Publication Title
Chem-seq permits identification of genomic targets of drugs against androgen receptor regulation selected by functional phenotypic screens.
Sample Metadata Fields
No sample metadata fields
View Samples